**7.1. Basal levels of MICA in different species**

62 New Approaches to the Study of Marine Mammals

images of Acetone and the three PAH treatments.

images of Ethanol and the four BPA treatments.

while it gives information on CYP1A1 inductive capacity (Figure 6 A-B). To be highlighted

**Figure 5.** A-E: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-E) in fibroblast cells of Bryde's whale (MBE3) (A), sperm whale (PM6) (B), Risso's dolphin (GGL1) (C), Striped dolphin (RT1) (D) and longbeaked common dolphin (MDC12) (E) treated with PAHs. DAPI and Alexa Fluor 594 (Intensity 200ms)

**Figure 6.** A-B: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-B) and CYP2B (C-D) in fibroblast cells of killer whale (MOO12) (A, B) treated with BPA. DAPI and Alexa Fluor 594 (Intensity 200ms)

that the highest used dose (100 μg/ml) is 100% lethal for fibroblast cells.

The basal level of MICA, evaluated with immunofluorescence technique in the fibroblasts of different cetacean species before treatment with different mixtures, is the first important result of this research step as it provides an indication of the immune status of these marine mammals. The results, expressed as immunofluorescence for cell (AUF/nucleus) mean values are presented in the Table 6 and, as histograms, in the Figure 7.


**Table 6.** Basal levels of immunofluorescence (AUF/nucleus) of MICA in fibroblast cells of Bryde's whale, long-beaked common dolphin, killer whale, sperm whale, bottlenose dolphin, striped dolphin and fin whale.

We can highlight that the three specimens belonging to the three species sampled in the Sea of Cortez (Bryde's whale, long-beaked common dolphin and killer whale) showed higher basal activity of MICA with respect to all Mediterranean specimens, regardless of the

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 65

**n° cells Mean Median Minimum Maximum S.D. S.E.** 

**n° cells Mean Median Minimum Maximum S.D. S.E.** 

**n° cells Mean Median Minimum Maximum S.D. S.E.** 

**n° cells Mean Median Minimum Maximum S.D. S.E.** 

of the immune system. The results of each specimen whose cells were treated with the two

**Killer whale** 

**BA** 98 195378 151675 42646 592187 162020 46176 **Inducer** 151 189172 202628 50932 350323 109320 30320

**0.8µg/ml** 135 106656 86680 46865 248599 61607 19481

**80µg/ml** 79 279350 250709 20631 636284 174779 50454.4 **Bottlenose dolphin** 

**BA** 87 31473 30832 26302 39107 4196.4 1713.2 **Inducer** 72 19230 19319 9871 44949 10153 3210

**0.8µg/ml** 66 21673 19480 19637 55989 13259 4193

**80µg/ml** 71 20647 17264 10492 56499 13536 4280 **Striped dolphin**

**BA** 75 18511 17896 8825 42083 9254 3055 **Inducer** 76 17606 18690 8556 26951 5977 1890

**0.8µg/ml** 69 20677 124525 41662 329043 13440 4250

**Fin whale** 

**BA** 114 31325 25172 18459 63324 16085 5086 **Inducer** 244 17409 17867 9673 23938 4270 1350

**0.8µg/ml** 78 28304 29231 13275 37051 69853 21061

**80µg/ml** 35 19031 18254 7299 56686 14102 4252

In all cases, with the exception of fibroblasts of killer whale treated with the highest dose of repressor and fibroblasts of striped dolphin treated with the lower dose of repressor (highlighted in dark grey), we can see a decrease of the response of the MICA compared to

**Table 7.** Descriptive statistics of immunofluorescence per cell (AUF/nucleus) of MICA revealed in cultured fibroblasts of Bryde's whale (MBE3), striped dolphin (RT23), bottlenose dolphin (TTA1), and killer whale (OO12) treated with the inducer (β-glucan) and the repressor (Cyclosporine A). BA represents the blank (cultured fibroblasts treated only with primary and secondary antibodies).

**80µg/ml All cell death** 

compounds are showed in Table 7.

**Repressor** 

**Repressor** 

**Repressor** 

**Repressor** 

**Repressor** 

**Repressor** 

**Repressor** 

**Repressor** 

**Figure 7.** Basal levels of immunofluorescence (AUF/nucleus) of MICA in fibroblast cells of Bryde's whale (B.e), long-beaked common dolphin (D.c.), killer whale (O.o.), sperm whale (P.m.), bottlenose dolphin (T.t.), striped dolphin (S.c.) and fin whale (B.p.).

species and the fact that they were sampled free-ranging or found stranded alive and then died. Moreover, the basal activity of MICA in the three Mexican species seems to be related to their different diet, with an increasing activity with the increase of the trophic level. Bryde's whale is not strictly planktophagus as is the fin whale in the Mediterranean, feeding mainly on blue fish. So this species in the food chain is definitely closer to long-beaked common dolphin and the other toothed whales in this study than to the other mysticete species. The free-ranging specimen of sperm whale sampled in the surrounding water of Asinara Island (Mediterranean Sea) showed a basal activity very similar to that showed by the striped dolphin, but lower than the other species sampled in the Mediterranean Sea (bottlenose dolphin and fin whale).

This sharp distinction between the activity of MICA found in the Sea of Cortez and Mediterranean Sea specimens is probably the most important result to be highlighted: it seems that the environment in which specimens live and, therefore, the anthropogenic stress to which they are subjected are determinant in the response of this protein. In the light of this result we can hazard the conclusion that the lower the anthropic stress of the specimens, the higher the basal activity of MICA. Regarding the Mediterranean species, the two stranded specimens (striped dolphin RT23 and fin whale RT25), were both affected by *morbillivirus*; it would be very interesting to know the basal activity in the same species sampled free-ranging, to understand whether in case of immunosuppression the activity of MICA increases or decreases. To assess whether MICA increases or decreases, because of the presence of an inducer or a repressor of the immune system, we treated fibroblast cell cultures with cyclosporine A (CsA), a drug that belongs to the category of immunosuppressants, and with β-glucan, a polysaccharide known to increase the response


of the immune system. The results of each specimen whose cells were treated with the two compounds are showed in Table 7.

64 New Approaches to the Study of Marine Mammals

dolphin (T.t.), striped dolphin (S.c.) and fin whale (B.p.).

(bottlenose dolphin and fin whale).

**Figure 7.** Basal levels of immunofluorescence (AUF/nucleus) of MICA in fibroblast cells of Bryde's whale (B.e), long-beaked common dolphin (D.c.), killer whale (O.o.), sperm whale (P.m.), bottlenose

species and the fact that they were sampled free-ranging or found stranded alive and then died. Moreover, the basal activity of MICA in the three Mexican species seems to be related to their different diet, with an increasing activity with the increase of the trophic level. Bryde's whale is not strictly planktophagus as is the fin whale in the Mediterranean, feeding mainly on blue fish. So this species in the food chain is definitely closer to long-beaked common dolphin and the other toothed whales in this study than to the other mysticete species. The free-ranging specimen of sperm whale sampled in the surrounding water of Asinara Island (Mediterranean Sea) showed a basal activity very similar to that showed by the striped dolphin, but lower than the other species sampled in the Mediterranean Sea

This sharp distinction between the activity of MICA found in the Sea of Cortez and Mediterranean Sea specimens is probably the most important result to be highlighted: it seems that the environment in which specimens live and, therefore, the anthropogenic stress to which they are subjected are determinant in the response of this protein. In the light of this result we can hazard the conclusion that the lower the anthropic stress of the specimens, the higher the basal activity of MICA. Regarding the Mediterranean species, the two stranded specimens (striped dolphin RT23 and fin whale RT25), were both affected by *morbillivirus*; it would be very interesting to know the basal activity in the same species sampled free-ranging, to understand whether in case of immunosuppression the activity of MICA increases or decreases. To assess whether MICA increases or decreases, because of the presence of an inducer or a repressor of the immune system, we treated fibroblast cell cultures with cyclosporine A (CsA), a drug that belongs to the category of immunosuppressants, and with β-glucan, a polysaccharide known to increase the response

**Table 7.** Descriptive statistics of immunofluorescence per cell (AUF/nucleus) of MICA revealed in cultured fibroblasts of Bryde's whale (MBE3), striped dolphin (RT23), bottlenose dolphin (TTA1), and killer whale (OO12) treated with the inducer (β-glucan) and the repressor (Cyclosporine A). BA represents the blank (cultured fibroblasts treated only with primary and secondary antibodies).

In all cases, with the exception of fibroblasts of killer whale treated with the highest dose of repressor and fibroblasts of striped dolphin treated with the lower dose of repressor (highlighted in dark grey), we can see a decrease of the response of the MICA compared to the basal activity. After the treatment with these doses, it therefore remains difficult to understand the response of MICA which we must expected from a toxicological stress. Probably the choice of the two compounds, known to have such capabilities in relation to the immune system but not specifically in respect to MICA, should be reassessed.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

*0.01 µg/ml 0.01 µg/ml 0.05 µg/ml 0.1 µg/ml* 

*0.1% Dose C Dose B Dose A* 

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 67

Only the sperm whale demonstrated a clear inductive effect on MICA due to OCs, compared to DMSO (Figure 8A). In fibroblasts of killer whale (MOO12) and striped dolphin (RT23) no dose caused an increase of the MICA with respect to the carrier (DMSO). A dose dependent induction of MICA was present in sperm whale (PMAS1), a bell-shaped response was present in Bryde's whale (MBE3) and in long-beaked common dolphin (MDC12) while

In the Table 9 are reported the results of the mean levels of immunofluorescence of MICA, revealed in cultured fibroblasts of different species treated with the flame retardants,

**MBE3 (Bryde's whale)** 100 49 35 55

**MOO12 (killer whale)** 100 66 54 96 **PMAS1 (sperm whale)** 100 102 74 102 **RT23 (striped dolphin)** 100 103 132 107 **RT25 (fin whale)** 100 101 134 120

**MDC12 (long-beaked common dolphin)** 100 69 96 209

**Table 9.** Mean values of immunofluorescence of MICA revealed in cultured fibroblasts of different species treated with flame retardants. The immunofluorescence is expressed in index numbers respect

In striped dolphin (RT23) and fin whale (RT25) the highest response of MICA was related to flame retardants, with a bell-shaped response in both species. Discontinuous induction response is shown by sperm whale (PMAS1) (Figure 8B) and long-beaked common dolphin (MDC12), while Bryde's whale (MBE3) and killer whale (MOO12) showed no induction

In Table 10 are reported the results of the mean levels of immunofluorescence of MICA, revealed in cultured fibroblasts of different species treated with the PAHs, expressed as

**MDC12 (long-beaked common dolphin)** 100 68 93 55

**Table 10.** Mean values of immunofluorescence of MICA revealed in cultured fibroblasts of different species treated with PAHs. The immunofluorescence is expressed in index numbers respect to solvent

**PMAS1 (sperm whale)** 100 101 181 189

to solvent control. Different colour of box is related to different increase of this protein.

**7.4. MICA in different species after treatment with PAHs** 

*MICA Acetone* 

control. Different colour of box is related to different increase of this protein.

discontinuous induction response was showed by fin whale (RT25).

*MICA Nonane* 

expressed as index numbers.

response.

index numbers.

**7.3. MICA in different species after treatment with Flame Retardants** 
